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METHODOLOGY - Coggle Diagram
METHODOLOGY
Soil treatment with 2,4-D butyl ester
- control pots only received distilled water and this must be done at least three replicates.
- the pots were sealed and placed in environment chamber at 25 C air temperature and 50% relative humidity in dark.
- after that, n 2,4-D butyl ester at concentration 10,100 or 100 microg g^1 soil was added to the treated pots
- the soil moisture was maintained at 60% water holding capacity with deionized water by doing aeration once a day.
- then, 2,4-D butyl ester were dissolved with water at concentration 200,2000 or 20000 0 microg ml^1
- Lastly, the pots were taken out from environment chamber randomly on 1,8,15,22 and 29 days for culturable microbial analysis(cfu) and 29
days for microbial community analysis (PLFA).
- 7 pots contain 10cm and 100g dry weight of soil samples were prepared and pre-incubated for 14 days in dark( 25 C)
SOIL SAMPLING
- two soil samples(no pesticides treatment) were collected from the field of the Institute of Plant Protection, China.
- the field then was divide into a grid and randomly sample. the soil samples from the top 0-15 cm were collected with a stainless steel soil tube drill with a diameter.
- after that, the duplicate composite sample,fully packed and label were taken into laboratory.
- the soil samples were mixed, then sieve with 2mm mesh so that the plant tissue will be removed.
- Divide the soil into two samples,soil 1 and soil 2 and and loaded with different water content, 14.3% and 13.7% respectively.
- then, the samples were pre-incubated for 14 days in the dark at 25 degree celcius to maintan water content.
- lastly, 2,4-D
butyl ester was added.
STATISTICAL ANALYSIS
- Significant differences p < 0.05, p < 0.01 , P <.001 * were accepted.
- The values of the three replicates are mean values and the the data in a column (or row) followed by the same letter do not differ significantly.
- The main component analysis (PCA) was used to examine the structure of the PLFA community among different samples which contained multiple variables.
- Lastly, the statistic were computed using Statistica 6.0 (Statsoft, Tulsa, OK, United States).
Microbial analysis
- For PLFA analysis, 5 g sample of freeze-dried soil was extracted with a onephase mixture of CHCl3/MeOH/citric acid buffer (0.15 M, pH 4)
(1:2:0.8, v/v/v).
- The lower layer of CHCl3 was collected and dried
under N2 for lipid fractionation.
- After that, the plates were incubated at 28 C and CFU can be counted after 5 days for bacteria, 7 days for fungi and 28 days for actinomycetes.
- After that, the extracted lipid were divided on silica gel columns into glycolipids, neutral lipids,
and polar lipids.
- then, 1ml of diluted sample was spread onto agar plates with beef extract-peptone medium.
- Using methanol KOH, polar lipids were transesterified to recover PLFAs as methyl esters in hexane by methanolysis.
- To get the CFU of bacteria,10mg of soil sample was suspended in 90ml sterile water and then was diluted by 10-fold serially dilution.
- The hexane supernatant containing the resulting fatty acid methyl esters (FAMEs) has been separated, quantified and identified using mass spectrometry (GCeMS) for gas chromatography.
9.PolarisQ ion trap GCeMS with HP-5ms column (60 m 0.25 mm inner diameter, 0.25 mm film thickness) has been used to identify FAME.
- 12 fatty acids 14:0, 2OH14:0, i15:0, a15:0, 15:0,
i16:0, 16:1u7c, 16:1u9c, 2OH16:0, i17:0, cy17:0, and cy19:0 was used to determine the bacterial biomass.
- The branched phospholipids i15:0, i16:0 and a15:0 were used as Gram-positive (GP) markers, while the PLFAs 16:1u7c, cy17:0 and cy19:0 were labeled Gram-negative (GN) bacteria.
- After that, The GN / GP ratio was used as a proxy for differences in the relative abundance of these microbial groups[1].
- Then,the microbial population stress level (cyc / precursor) was determined from the ratio of (cyc17:0 to cyc19:0)/(16:1u7c to 18:1u7c)
- Lastly, the result will be obtain as the percentage of the total PLFA.